Phytochemical and biological investigation of Astragalus Caprinus L

Background cultivated and wild plants are used to treat different ailments. The Astragalus genus is found in temperate and dry climates; thus, it is found in Egypt and the arab world. Astragalus caprinus has a good amount of bioactive chemicals, which may help explain its therapeutic effects in reducing the risk of consequences from disease. Method The phytochemical investigation of the herb and roots of Astragalus caprinus L. included the analytical characterization for the petroleum ether components by GC/MS, unsaponifiable matter (unsap. fraction), and fatty acids (FAME) investigation by GLC analysis. Main flavonoids were chromatographically isolated from ethyl acetate and n-butanol extracts. In vitro antimicrobial activity has been tested against the Gram-positive bacteria Staphylococcus aureus and Streptococcus mutans for different plant extracts, the Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella pneumonia, the fungus Candida albicans and Aspergillus niger, and the Escherichia coli bacterium. Metabolite cytotoxicity was examined using the MTT assay against HepG-2 (human liver carcinoma) and MCF-7 (breast carcinoma). Results Identifying the important components of the herb and root petroleum ether extracts was achieved. Using column chromatography, luteolin, cosmosiin (apigenin-7-O-glucoside), and cynaroside (luteolin-7-O-glucoside) were separated and identified using UV, NMR, and Mass Spectroscopy. Root extracts displayed potential antimicrobial activity against most of the tested pathogens. Both extracts (herb and roots) were active against the MCF-7 cell line and HepG-2 cell line with IC50 62.5 ± 0.64 and 72.4 ± 2.3 µg/ml, and 75.9 ± 2.5 and 96.8 ± 4.2 µg/ml, respectively. Conclusion Astragalus caprinus seems to be a promising source of bioactive compounds that could potentially aid in preventing disease complications and address common health issues in developing countries. Moreover, the various parts of this plant could be utilized as natural raw materials for producing health-boosting products that could address common health issues in developing countries.


Introduction
Astragalus is one of the largest genera in the plant family Fabaceae, which is widely distributed throughout the temperate and arid regions of the world [1].Astragalus species growing in North Africa are Mediterranean or Arabian Saharan plants [2,3].Astragalus species have long been used in folk medicine against stomach ulcers, chronic bronchitis, hypertension, and diabetes [1].Secondary metabolites are very important for their low cost and high biological activity [4].Among the significant bioactive secondary metabolites found in Astragalus species are triterpenes, fatty acids, polysaccharides, saponins, flavonoids, and alkaloids [5][6][7][8][9].Consequently, the genus possesses engaging pharmacological activities, including immunoregulatory, antitumor, antidiabetic, antioxidative, and antimicrobial, and is also considered cardioprotective [10][11][12].
Astragalus caprinus is a common species in the Fabaceae family widely distributed in northwest Africa.It extends from the northern Mediterranean region over several climatic zones, native to Egypt [13,14].Flavonoids were extracted and analyzed from air-dried leaves [15][16][17].We examined the seeds' lipid content, fatty acid composition, proteins, and bioactive substances [18].
In developing countries, drugs are considered to be very expensive for many people to purchase nowadays.Researchers should evaluate the endogenous plant flora of each region to obtain more data on the chemical and biological significance of indigenous plants.Egypt and the Arab world are home to the Astragalus genus, which can be found in temperate and dry climates.The presence of bioactive chemicals in Astragalus caprinus could explain its therapeutic effects in reducing the risk of disease consequences.
This study is designed to assign a comparative chemical composition of the herb and roots of Astragalus caprinus subspecies (ssp.)langaraise through the analytical characterization for nonpolar components as well as saponifiable and unsaponifiable compounds by GC/MS and GLC, isolation of the significant flavonoidal constituents that may correlate with its pharmacological activities.Evaluating the antimicrobial activity against selected influential bacteria and fungi and its cytotoxic activity against MCF-7 (breast carcinoma) and HepG-2 (human liver carcinoma) in vitro using MTT assay.

Plant material
Astragalus caprinus (AC) was collected during spring and early summer (2020-2021) from El-Magtala between Mersa Matruh and Sidi Brani, Mediterranean coast, and graciously recognized by Dr. Mohammed Elgebaly, a professor at the National Research Center's (NRC) Department of Phytochemistry and Plant Systematics.A voucher specimen was placed in the NRC Herbarium (No.209).The roots were separated from the herb and then dried in an oven at 40 o C.After drying, the herb and roots were separately ground to a fine powder.

Extraction and isolation of lipid constituents
The powdered herbs and roots (H and R) (500gm) were individually extracted in a soxhlet device eachusing petroleum ether (b.r.40-60 °C).Fuller's earth was run through the H extract to eliminate the colorful pigments.Both extracts (H and R) were filtered, dried over anhydrous sodium sulphate, and then evaporated in a vacuum at 40 °C until completely dry, yielding 1.6% and 1.1% v/w, respectively, of yellow, oily residues that were refrigerated until their GC/MS analysis.About 5 g of each oily residue was individually submitted to the saponification process to get the saponifiable and unsaponifiable fractions for the lipid content research [19].Following saponification, the free fatty acids were methylated using the procedure outlined by Zargoun [20] to produce free acid methyl esters (FAME).

GC/MS analysis of petroleum ether extract
The analysis of petroleum ether extract of H and R was carried out utilizing a capillary gas chromatography type Trace GC ULTRA from (Thermo Scientific) directly coupled to ISQ Single Quadruple MS and equipped with TG-5MS, nonpolar 5% phenyl methylpolysiloxane capillary column (30 m × 0.25 mm ID × 0.25 μm).The operating conditions were applied as reported by Zargoun [20].

Gas-liquid chromatographic analysis of lipid fraction
The unsaponifiable matter of both H and R was analyzed using Agient Technologies' 6890 N Network GC system, according to Abdelshafeek [21].

Extraction and isolation of flavonoids
To separate the flavonoids, the defatted H marc was extracted using 80% MeOH (5 L x 3) for three days at room temperature until complete exhaustion using percolator.Filtration and concentration under reduced pressure gave the hydrophilic residue, which was dissolved in H 2 O and then partitioned successively with CH 2 Cl 2 , EtOAc, and n-BuOH.The different extracts were concentrated and screened using TLC.Compound 1 was obtained by chromatographing the ethyl acetate residue (5 g) on a silica gel column and then utilizing 100% methanol on a Sephadex LH-20 Column to separate the primary components.The n-butanol extract (6 g) was chromatographed on a polyamide column using a gradient of water/methanol (100:0 to 20:80).Fractions having similar PC profiles were pooled to afford ten fractions (F-1 to F-10).To obtain the pure isolated compounds (2-3), fractions F-5 and F-7 containing the essential compounds were repeatedly chromatographed on the Sephadex LH-20 column and eluted with various gradients of methanol/water.

Antimicrobial assay
The antimicrobial activity of the successive extracts of both H and R were separately tested in vitro for their antibacterial activity using the agar well diffusion method against the Gram-positive bacteria, Staphylococcus aureus (ATCC: 13,565) and Streptococcus mutans (ATCC: 25,175), and the Gram-negative bacteria; Escherichia coli (ATCC: 10,536) and Klebsiella pneumonia (ATCC: 10,031) using nutrient agar medium.The antifungal activity was tested against Candida albicans (ATCC: 10,231) and Aspergillus niger (ATCC: 16,404) using Sabouraud dextrose agar medium [22].Standard treatments for Gram-positive and Gram-negative bacteria were ampicillin and gentamicin, respectively.The standard treatment for fungal strains was the use of Nystatin.As a solvent (negative) control, DMSO was employed.The extracts were tested at a 15 mg/ml concentration against bacterial and fungal strains.This experiment was carried out in triplicate, and inhibition zones were measured in mm scale.

Statistical analysis
Every experiment was carried out three times.Graph-Pad Prism® v6.0 software (GraphPad Software Inc., San Diego, CA, USA) created the concentration-response curve and fit the non-linear regression model.All results were reported as mean ± SD.IC 50 s were determined by probit analysis using SPSS software program (SPSS Inc., Chicago, IL).

GC/MS analysis of petroleum ether extract
The chemical constituents of the petroleum ether extract of both H and R were identified using GC/MS analysis.Table 1 (Figs. 1 and 2) showed that the petroleum ether extracts of H and R of A. caprinus contained 34 and 35 compounds, respectively, belonging to different classes of phytoconstituents, including hydrocarbons which constitute 53.5% and 55.17%, respectively, with n-undecane as main one (14.32%and 11.83%, respectively), aromatic components (34.92% and 35.20% ) in which 5-phenyl undecane is the main compound (4.34% and 3,49%, respectively), only one monoterpene hydrocarbon (pmenthane) in both H and R (2.35% and 1.69%).Sesquiterpene hydrocarbons are present only in H (1.77%) and absent in R, in addition to acids, which are absent in H and present in R (2.12%).To our knowledge, these data are reported for the first time for A. caprinus ssp.langarise, Table 2; Fig. 3 shows the percentage of the different compound classes of the petroleum ether extract of H and R. Considering other species, such as A. sieberi, a study revealed the presence of four compounds from the pet.ether fraction with N, N-dimethyl-1-Dodecanamine (42.36%), and butylated hydroxytoluene (35.96%) as major compounds detected in the GC/MS analysis constituting 78.32% of the total peak area [26].B-sitosterol and ceryl alcohol were isolated from the unsaponifiable fraction of Astragalus cremophilos, and the fatty acids were studied by GLC [27].

GLC analysis of unsaponifiable fraction
The unsaponifiable matter of both H and R was examined using GLC.The total ion chromatograms are displayed in Figs. 4 and 5 and the components are shown in Table 3.The GLC analysis of H revealed a combination of triterpenes, sterols, and hydrocarbons.The predominant hydrocarbon was C 14 (32.98%), with the other hydrocarbons ranging from C 11 to C 30 .Campesterol was the predominant sterol (1.62%), and only one triterpene (β-amyrin) was detected.The GLC analysis of R revealed the presence of a mixture consisting of sterols and hydrocarbons in which the predominant hydrocarbon is C 25 (54.16%), with the other hydrocarbons ranging from C 11 to C 30 .Campesterol was also the predominant sterol (5.67%).
Figure 6 Displays the hydrocarbon percentage of H and R.

GLC analysis of fatty acid methyl esters
The data of the GLC analysis of the fatty acid methyl esters of both H and R (Figs. 7 and 8) displayed nine fatty acids in H, accounting for 94.25 of the total acid percentage.The primary unsaturated fatty acids are linolenic acid (33.53%) and linoleic acid (15.15%), while the major saturated fatty acid is palmitic acid (24.90%).The results of R revealed the presence of eight fatty acids, representing 99.46% of the total.Linolenic acid was the main one (23.11%),followed by Linoleic acid (22.05%).The major saturated fatty acid was palmitic acid (21.03%), as shown in Table 4. Figure 9 shows the percentage of unsaturated fatty acids in H and R.

Identification of flavonoids
Compound 1 luteolin, this compound was isolated as an amorphous yellowish powder, and it exhibited band-I in the UV spectrum with methanol at λ max = 347 nm, which proves its flavone nature.The presence of an ortho dihydroxy system was confirmed through AlCl 3 /HCl spectrum, where there is a hypthochromic shift (34 nm) in the band-I relative to AlCl 3 spectrum.The MS spectrum gave M + at m/z = 286, corresponding to the molecular formula C 15 H 10 O 6 [30].
Compound 2 After re-chromatography, cosmosiin (apigenin-7-O-glucoside) was isolated as a pale yellow powder.The UV spectra of the compound in methanol and different shift reagents confirmed its flavone nature, characterized by band-I at λmax = 330nm, and the absence of an ortho dihydroxy system.The acid hydrolysis of the compound resulted in the formation of apigenin as an aglycone and glucose as a sugar.The attachment of the sugar moiety at C-7 was confirmed through band-2 at λmax = 274 nm in the sodium acetate spectrum relative to  ).The anomeric proton of the glucose appeared at 5.39 (d, J = 6.9 Hz, H-1") and δ C at 99.87 (C-1").The other data obtained were consistent with previously reported data [30].
Compound 3 cynaroside (luteolin-7-O-glucoside) was isolated from the butanol fraction after column chromatography and obtained as a yellowish amorphous powder.Its chromatographic behavior and acid hydrolysis substantiate its glycosidic nature with only one glucose moiety.The UV spectra showed absorption maxima of band-I in methanol at λ max = 348 nm, which proved its flavone nature; also, it established the absence of a free OH group at C-7 with no bathochromic shift in band-II (260 nm) of sodium acetate spectrum relative to methanol spectrum (256 nm).The EI-MS spectrum showed the molecular ion peak M + as a small peak at m/z = 448,  [30].The chemical structure of isolated flavonoids is presented in Fig. 10.

Antimicrobial activity
The growth absence of the microorganism (clear zone) and the diffusion of an antibiotic agent in the medium after 24 h for evaluation are related to the inhibitory zone in the well diffusion method.As indicated in Table 5, the investigation revealed that the subsequent extracts of both the H and R demonstrated differing degrees of activity against the strains that were examined, except for the Gram-negative bacteria Klebsiella pneumonia, the Gram-positive bacteria Streptococcus mutans, and the fungus Aspergillus niger.

Cytotoxic assay
The in vitro cytotoxicity of A. caprinus 80% MeOH extracts of H and R against HepG-2 and MCF-7 human carcinoma cell lines was evaluated using an MTT assay with Doxorubicin as a positive control.The obtained results are given in Table 6.

Discussion
Many natural compounds with little or no reported adverse effects have demonstrated considerable health benefits and are found in the kingdom of plants.These therapeutic plants are abundant in phytochemical substances with critical therapeutic applications for treating various metabolic disorders.According to Tungmunnithum [31], epidemiological research has linked plant phenolics to a protective effect that lengthens average life spans and reduces the prevalence of a wide range of  human disorders.Astragalus species are small shrubs or herbs with stems that develop from underground roots, either annual or perennial.These priceless plants are used as fuel, food, medicine, fodder, and decorative.Astragalus L. plants are of tremendous importance as sources of active compounds, as evidenced by the numerous studies conducted on them, including saponins, amino acids, polysaccharides, flavonoids, glycosides, organic acids, and alkaloids that have promising biological potentials [32][33][34].It helps treat disorders of the urinary, respiratory, metabolic, digestive, and neurological systems and issues with the blood, circulatory system, and skin.It can be taken as a decoction, chewing, infusion, poultice, or powder [35][36][37].
In the present investigation, quantitation of the petroleum ether extract of H and R of A. caprinus was done by GC/MS.The extract contained 34 and 35 compounds, respectively, belonging to different classes of phytoconstituents, including hydrocarbons and aromatic components.Sesquiterpene hydrocarbons were present only in H and absent in R, in addition to acids, which are absent in H and present in R. The main components of the unsaponifiable fraction and the majority of the fatty acid methyl esters for both extracts were also identified.The GLC analysis of H revealed a combination of triterpenes, sterols, and hydrocarbons, while R revealed a mixture of sterols and hydrocarbons.The data of the GLC analysis of the fatty acid methyl esters of both H and R showed that the main unsaturated fatty acids were linolenic acid and linoleic acid.In contrast, the major saturated fatty acid was palmitic acid, which, following the previous study [29] and the development of novel anti-infective drugs, have represented an active research area.
According to earlier research, it may provide some health benefits because of the flavonoids it contains.The extracts of A. membranaceus roots from four different origins were examined by UHPLC-MS/MS using principal component analysis, and eighteen compounds were classified as major components, including apigenin 7-O-glucoside.Recently, three flavonoids were isolated and identified from the aqueous methanol extract, namely, luteolin, cosmosiin (apigenin 7-O-glucoside), and cynaroside, representing flavone aglycone and flavone glycosides.
The discovery of natural compounds has been recognized as a rich source of these drugs.With this, we demonstrated the antimicrobial activity of Astragalus caprinus in different extracts of the herb and roots.Our results revealed that the polar extracts (aqueous, methanol, and n-butanol) were more effective against the tested  organisms, and the root extracts were considerably found to be broadly effective against the pathogens.This activity may be related to the high concentration of flavonoids and phenolic compounds in the extract, which act as antimicrobial agents through numerous mechanisms [9,38,39].They provide opportunities for innovation in drug discovery because both monophenolic and polyphenolic compounds from various plants have been demonstrated to inhibit, block, or attenuate the initiation, progression, and spread of cancer in normal or pre-neoplastic tissues.They have also been shown to interfere with different stages of carcinogenesis in vitro and in vivo studies through a process known as radical scavenging.
It's interesting to note that the scientific community has been closely studying Astragalus species due to their potential for cytotoxicity.Recent research has shown that the genus possesses intense anticancer activity, which is relevant to its cytotoxic potential [40].One particular study found that Astragalus plants exhibited cytotoxic ability against MCF-7 and HepG-2 cancer cell lines, indicating the function of its phenolic chemicals [12,41].It's worth noting that more research is necessary to search for new anticancer drugs.This present scientific study has gathered data that shows Astragalus caprinus is high in bioactive polyphenols that may be responsible for various pharmacological activities.As a result, it needs to be further investigated for its potential uses as a natural health-promoting agent.

Conclusion
Astragalus caprinus seems to be a promising source of bioactive compounds that could potentially aid in preventing disease complications.Moreover, the various parts of this plant could be utilized as natural raw materials for producing health-boosting products that could address common health issues in developing countries.
Further toxicity investigation is recommended for this plant to be one of the promising plants in the pharmacological industry.

Fig. 2 Fig. 1
Fig. 2 GC chromatogram of petroleum ether extract of R

Fig. 5
Fig. 5 GLC chromatogram of Unsaponifiable fraction of the R

Fig. 7 Fig. 6
Fig. 7 GLC chromatogram of fatty acid methyl esters of H

Fig. 8
Fig. 8 GLC chromatogram of fatty acid methyl esters of R

Fig. 10
Fig. 10 Chemical structure of isolated flavonoids

Fig. 9 Table 5
Fig. 9 Percentage of unsaturated fatty acids in H and R

Table 1
GC/MS data of petroleum ether extract of both H and

Table 2
Classes of Pet.ether ext of both H and R

Table 3
GLC data of Unsaponifiable fractions of both H and R

Table 4
GLC data of fatty acid methyl esters of H and R

Table 6
The IC 50 values of H and R methanol extracts using MTT assay